摘要
用大黄鱼Wap65-2基因成熟肽区构建酵母双杂交诱饵质粒,并对诱饵质粒进行自激活活性及毒性检测。从大黄鱼(Larimichthys crocea)肝组织c DNA中扩增Wap65-2基因成熟肽片段(Lc Wap65-2m),将其克隆至p GBKT7载体中,获得诱饵载体p GBKT7-Lc Wap65-2m,经PCR、酶切和测序验证无误后,转化至酵母菌株Y187中,在营养缺陷培养基中观察重组蛋白的毒性作用和自激活活性,同时利用Western blot分析重组蛋白的表达。成功扩增Lc Wap65-2m,并克隆至p GBKT7载体中;Western blot检测结果表明,p GBKT7-Lc Wap65-2m在酵母细胞中表达诱饵蛋白Lc Wap65-2m;表型筛选结果显示,Lc Wap65-2m无毒性作用和自激活活性。成功构建了Lc Wap65-2m酵母双杂交诱饵载体,为进一步利用酵母双杂交技术筛选与Wap65-2相互作用的蛋白奠定基础。
The mature peptide of Wap65-2 gene （ LcWap65-2m ） in large yellow croaker （ Larimichthys crocea ） was used to construct the yeast two-hybrid bait vector, and its self-activation and toxic effect were measured. LcWap65-2m from the liver cDNA of large yellow croaker was amplified, and cloned into the plasmid pGBKT7. After verification by PCR amplification, enzyme digestion and sequencing, the bait vector pGBKT7-LcWap65-2m was transformed into the yeast strain Y187. The self-activation and toxic effect of the recombinant protein were detected in selective nutrition medium, and the expression of the bait protein LcWap65-2m was analyzed by Western blot. LcWap65-2m was successfully amplified and inserted into the plasmid vector pGBKTT. Western blot analysis showed that the bait protein LcWap65-2m was expressed in yeast cells ; phenotypic screening tests revealed that the bait protein LcWap65-2m had no self-activation activity and toxic effect. In conclusion, the yeast two-hybrid bait vector pGBKTT-LcWap65-2m was successfully constructed, which laid the foundation for screening the protein interacted with the bait protein Wap65-2 using the yeast two-hybrid system.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第8期108-113,共6页
Biotechnology Bulletin
基金
国家自然科学基金项目(31402333)
高等学校博士学科点专项科研基金项目(20133305120008)
宁波市自然科学基金项目(2013A610160)
浙江省教育厅科研项目(理)(Y201327619)
